Insulin production methods and pro-insulin constructs

ABSTRACT

Novel pro-insulin having specific amino acid and/or nucleic acid modifications suitable for improved methods of insulin production are provided. Novel and highly efficient processes for preparing the pro-insulin preparations and preparations containing them are also disclosed. The novel pro-insulin preparations may be converted into human insulin useful in therapeutic preparations. Novel peptides of the C-peptide, and N terminus, including RREAEALQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO:32), and MHHHHHHGGR (SEQ ID NO:36) respectively are provided, as well as the unique nucleic acid molecules encoding them.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/725,731, filed Mar. 20, 2007, which claims priority to andthe benefit of U.S. Provisional Patent Application Ser. No. 60/874,655,filed Dec. 13, 2006. The entire disclosure and contents of thesedocuments are hereby incorporated by reference.

BACKGROUND

1. Field of the Invention

The present invention in a general and overall sense relates to thefield of recombinant proteins and peptides. The invention also relatesto the field of molecular processes and methods for producingrecombinant proteins particularly methods that employ E. coli as anexpression vehicle. The invention also relates to compositions andmethods for preparing pro-insulin, insulin, and both of these alone orin combination with each other and with other compositions.

2. Background of the Related Art

Insulin is a polypeptide hormone secreted by beta-cells of the pancreas.This hormone is made up of two polypeptide chains, an A-chain of 21amino acids, and a B-chain of 30 amino acids. These two chains arelinked to one another in the mature form of the hormone by twointerchain disulfide bridges. The A-chain also features one intra-chaindisulfide bridge.

Insulin is a hormone that is synthesized in the body in the form of asingle-chain precursor molecule, pro-insulin. Pro-insulin is a moleculecomprised of a prepeptide of 24 amino acids, followed by the B-chainpeptide, a C-peptide of 35 amino acids, and an A-chain peptide. TheC-peptide of this precursor insulin molecule contains the two aminoacids, lysine-arginine (LR) at its carboxy end (where it attaches to theA-chain), and the two amino acids, arginine-arginine (RR) at its aminoend (where it attaches to the B-chain). In the mature insulin molecule,the C-peptide is cleaved away from the peptide so as to leave theA-chain and the B-chain connected directly to one another in its activeform.

Molecular biology techniques have been used to produce humanpro-insulin. In this regard, three major methods have been used for theproduction of this molecule. Two of these methods involve Escherichiacoli, with either the expression of a large fusion protein in thecytoplasm (Chance et al. (1981), and Frank et al. (1981) in Peptides:Proceedings of the 7^(th) American Peptide Chemistry Symposium (Rich, D.and Gross, E., eds.), pp. 721-728, 729-739, respectively, PierceChemical Company, Rockford, Ill.), or the use of a signal peptide toenable secretion into the periplasmic space (Chan et al. (1981)P.N.A.S., U.S.A., 78:5401-5404). A third method utilizes yeast,especially Saccharomyces cerevisiae, to secrete the insulin precursorinto the medium (Thim, et al. (1986), P.N.A.S., U.S.A., 83: 6766-6770).

Chance et al. report a process for preparing insulin by producing eachof the A and B chains of insulin in the form of a fusion protein byculturing E. coli that carries a vector compromising a DNA encoding thefusion protein, cleaving the fusion protein with cyanogen bromide toobtain the A and the B chains, sulfonating the A and B chains to obtainsulfonated chains, reacting the sulfonated B chain with an excess amountof the sulfonated A chain; and then purifying the resultant products toobtain insulin. Drawbacks associated with this process are that itrequires two fermentation processes and the requirement of a reactionstep for preparing the sulfonated A chain and the sulfonated B chain.This results in a low insulin yield.

Frank et al. described a process for preparing insulin in the form of afusion protein in E. coli. In this process, pro-insulin is produced inthe form of a fusion protein by culturing E. coli which carries a vectorcomprising a nucleic acid sequence (DNA) encoding for the fusionprotein, cutting the fusion protein with cyanogens bromide to obtainpro-insulin, sulfonating the pro-insulin and separation of thesulfonated pro-insulin, refolding the sulfonated pro-insulin to formcorrect disulfide bonds, treating the refolded pro-insulin with trypsinand carboxypeptidase B, and then purifying the resultant product toobtain insulin. However, the yield of the refolded pro-insulin havingcorrectly folded disulfide bonds is reported to sharply decrease as theconcentration of the pro-insulin increases. This is allegedly due to, atleast among other reasons, misfolding of the protein, and some degree ofpolymerization being involved. Hence, the process entails theinconvenience of using laborious purification steps during the recoveryof pro-insulin.

Thim et al. report a process for producing insulin in yeast,Saccharomyces cerevisiae. This process has the steps of producing asingle chain insulin analog having a certain amino acid sequence byculturing Saccharomyces cerevisiae cells, and isolating insulintherefrom through the steps of: purification, enzyme reaction, acidhydrolysis and a second purification. This process, however, results inan unacceptably low yield of insulin.

The role of the native C-peptide in the folding of pro-insulin is notprecisely known. The dibasic terminal amino acid sequence at both endsof the C-peptide sequence has been considered necessary to preserve theproper processing and/or folding of the pro-insulin molecule to insulin.

Other amino acids within the C-peptide sequence, however, have beenmodified with some success. For example, Chang et al. (1998) (Biochem.J., 329:631-635) described a shortened C-peptide of a five (5) aminoacid length, -YPGDV- (SEQ ID NO:1), that includes a preserved terminaldi-basic amino acid sequence, RR at one terminal end, and LR at theother terminal end, of the peptide. Preservation of the dibasic aminoacid residues at the B-chain-C peptide (B-C) and C-peptide-A-chainjunctures is taught as being a minimal requirement for retaining thecapacity for converting the pro-insulin molecule into a properly foldedmature insulin protein. The production of the recombinant human insulinwas described using E. coli with a shortened C-peptide having a dibasicamino acid terminal sequence.

One of the difficulties and/or inefficiencies associated with theproduction of recombinant insulin employing a pro-insulin constructhaving the conserved, terminal di-basic amino acid sequence in theC-peptide region is the presence of impurities, such as Arg-insulin, inthe reaction mixture, once enzymatic cleavage to remove the C-peptide isperformed. This occurs as a result of misdirected cleavage of thepro-insulin molecule so as to cleave the C-peptide sequence away fromthe A-chain at this juncture, by the action of trypsin. Trypsin is atypical serine protease, and hydrolyses a protein or peptide at thecarboxyl terminal of an arginine or lysine residue (Enzymes, pp. 261-262(1979), ed. Dixon, M. & Webb, E. C. Longman Group Ltd., London). Thisunwanted hydrolysis results in the unwanted ARG-AO-insulin by-product,and typically constitutes about 10% of the reaction yield. Hence, anadditional purification step is required. The necessity of an additionalpurification step makes the process much more time consuming, and thusexpensive, to use. Moreover, an additional loss of yield may be expectedfrom the necessity of this additional purification step.

Others have described the use of pro-insulin constructs that do not havea conserved terminal dibasic amino acid sequence of the C-peptideregion. For example, U.S. Pat. No. 6,777,207 (Kjeldsen et al.) relatesto a novel pro-insulin peptide construct containing a shortenedC-peptide that includes the two terminal amino acids, glycine-arginineor glycine-lysine at the carboxyl terminal end that connects to theA-chain of the peptide. The B-chain of the pro-insulin constructdescribed therein has a length of 29 amino acids, in contrast to thenative 30 amino acid length of the native B-chain in human insulin. Thepotential effects of this change to the native amino acid sequence ofthe B-chain in the human insulin produced are yet unknown. Methods ofproducing insulin using these pro-insulin constructs in yeast are alsodescribed. Inefficiencies associated with correct folding of the matureinsulin molecule when yeast utilized as the expression host, render thisprocess, among other things, inefficient and more expensive and timeconsuming to use. In addition, yeast provides a relatively low insulinyield, due to the intrinsically low expression levels of a yeast systemas compared to E. coli.

As evidenced from the above review, a present need exists for a moreefficient process for production of human insulin that is efficient,eliminates currently necessary purification steps, and that at the sametime improves and/or preserves acceptable production yield requirementsof the pharmaceutical industry.

The above references are incorporated by reference herein whereappropriate for appropriate teachings of additional or alternativedetails, features and/or technical background.

SUMMARY OF THE INVENTION

The present invention provides novel pro-insulin compositions. Thesecompositions may be further defined as comprising a peptide, an aminoacid sequence, or a nucleic acid sequence encoding a modified C-peptideor a modified pro-insulin molecule, or a pharmaceutical preparation ofthese peptides in a pharmaceutically acceptable carrier solution and/ordiluent. Among other distinguishing features, the novel pro-insulin asdisclosed herein includes a unique non-di-basic amino acid sequence atone or both of its C-peptide terminal ends. In some embodiments, theconstructs are described as an N-terminal multiple His-taggedpro-insulin construct. In particular embodiments, the N-terminalmultiple His-tagged pro-insulin construct comprises a 6-histidineN-terminal tag (SEQ ID NO:24). In some embodiments, the constructcomprises a structure as defined in Formula I or Formula II:

Formula I: MHHHHHHGGR (SEQ ID NG:2)-X1-C peptide-X2, or Formula II:MHHHHHHGGR (SEQ ID NG:2)-X1-modified C peptide-X2wherein X1 comprises an insulin B Chain, and wherein X2 comprises aninsulin A Chain. In the Formula II construct, the modified C peptide isdefined as a sequence having a non-dibasic (such as AR) amino acidsequence at a terminal end of the modified C peptide component that isadjacent the X2 component (insulin A chain) and/or a modification (suchas RA) located adjacent the X1 component (insulin B chain) in theconstruct as depicted in Formula II.

The invention in particular aspects may be further defined as providinga novel pro-insulin composition. This composition may be further definedas comprising a peptide, an amino acid sequence, or nucleic acidsequence encoding the pro-insulin. The purified and transformedpro-insulin comprises a pharmaceutical preparation formulated with apharmaceutically acceptable carrier solution and/or diluent.

The invention also provides a novel process for producing highlypurified insulin that is more efficient than current techniques. Inparticular aspects, the process employs bacteria, such as E. coli.

The process presents many advantages, among them the advantage ofreducing and/or eliminating the presence of unwanted and contaminatingcleavage by-products characteristic of conventional manufacturingprocesses for producing recombinant human insulin in E. coli. Previouslyundesirable by-products evident in yield mixtures using conventionalmethods of producing recombinant human insulin included, by way ofexample, the production of an unwanted cleavage product, ARG-AO-insulin.A highly efficient process for the production of recombinant humaninsulin is presented that reduces and/or eliminates the presence of thisand other unwanted and undesirable cleavage by-products, and thatfurther presents the advantages of eliminating several time consuming,expensive, purification steps. A process having fewertechnician-assisted steps is thus devised, and illustrates theadditional advantage of eliminating the degree of inconsistency and/orerror associated with technician assisted steps in the manufacturingprocess.

In some embodiments, the preparations comprise a pharmaceuticallyacceptable preparation comprising recombinant human insulin and beingessentially free of pro-insulin.

An object of the invention is to solve at least the above problemsand/or disadvantages and to provide at least the advantages describedhereinafter.

Additional advantages, objects, and features of the invention will beset forth in part in the description which follows and in part willbecome apparent to those having ordinary skill in the art uponexamination of the following or may be learned from practice of theinvention. The objects and advantages of the invention may be realizedand attained as particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in detail with reference to thefollowing drawings in which like reference numerals refer to likeelements wherein:

FIG. 1, according to one aspect of the invention, presents a gel showingexpression of the full length His-tagged/K64A pro-insulin (10.5 KDa)protein. The gel is from the expression product produced from the E.coli, BL21 strain, transformed with the above mentioned genes.

FIG. 2, according to one aspect of the invention, presents an HPLC ofrecombinant IPT human insulin. The chromatograph demonstrates that thepreparation is high in purity with almost non-existent levels ofpro-insulin.

FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

N-Terminal 6-His tagged clone with tryptic cleavage sequence forremoval: (Utilization of a Nickel chelating column for high purity in asingle step)

(SEQ ID NO: 28) MHHHHHHGG RFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

Site Directed Mutagenesis of Lysine 64 to Alanine: Prevention ofincorrect cleavage following the lysine. No arg-insulin contaminantgenerated, resulting in an increased yield.

(SEQ ID NO: 29) MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ A RGIVEQCCTSICSLYQLENYCN

Example 9 Purification Method for Production of Lys/Pro Insulin

The present example demonstrates the utility of the present inventionfor providing a unique construct and purification scheme thatsignificantly improves the purification method for the production ofLys/Pro Insulin.

Lys/Pro Insulin is characterized as a short acting insulin analog,which, when combined with am insulin pump, allows for better bloodglucose stability without the risk of hyperglycemia.

Amino acid sequence of the unique construct as defined in example 8.

(SEQ ID NO: 30) MHHHHHHGG RFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ A RGIVEQCCTSICSLYQLENYCN

Lys/Pro insulin modification with residues 28 and 29 reversed in theirorder:

(SEQ ID NO: 31) MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYT KP TRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCN

A general outline of one method, by way of example and not exclusion, toisolate and/or enrich recombinant Lys/Pro insulin from a compositionthat is not enriched for FIG. 3, according to some aspects of theinvention, presents a biopotency study showing relative glucose curvesfor rabbits injected with saline (control), Humulin R (positivecontrol), and Elona Biotechnologies human insulin (test sample).

FIG. 4, according to some aspects to the invention, presents a flowscheme for the purification of insulin and Lys-Pro insulin, using thepurification methods described in examples 1 and 9.

FIGS. 5A and 5B, according to some aspects of the invention, presents areverse phase chromatography analysis of chromatogram with (5A) andwithout (5B) sodium sulfate.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides novel pro-insulin constructs, C-peptides,and methods for using these in a process to provide high yields ofmature recombinant human insulin. The pro-insulin constructs include anovel C-peptide that includes a non-dibasic terminal amino acid sequenceat one end.

In particular embodiments, the non-dibasic alanine-arginine (AR)sequence at the carboxyl terminus of the C-chain. The C-terminus of theC-peptide connects to the A-chain of the pro-insulin molecule.Advantageously, the positioning of these particular terminal amino acidsin the C-peptide provides for an improved method for producingrecombinant human insulin, having fewer steps, improved yields of therecombinant human insulin protein and less contaminating byproducts.

As used in the description of the present invention, the term“connecting peptide” or “C-peptide” is meant the connecting moiety “C”of the B-C-A polypeptide sequence of a single chain pro-insulinmolecule. Specifically, in the native human insulin chain, the C-peptideconnects to position 30 of the B-chain and position 1 of the A-chain. Inthe present invention, the A-chain and the B-chain of the pro-insulinconstructs retain their native sequences and lengths. The C-peptideconstructs disclosed have been modified so as to include differentterminal amino acids relative to native C-peptide.

As in native human pro-insulin, the C-peptide constructs of the presentinvention connect position 30 of the B-chain and position 1 of theA-chain. The single chain pro-insulin molecules of the invention willinclude three (3) correctly positioned, disulphide bridges, as ischaracteristic of the native human pro-insulin molecule. The amino acidsequence of the B-chain and the A-chain of the pro-insulin constructs,as well as the human insulin products produced by the methods describedherein, will have the native amino acid sequence characteristic ofnative human insulin.

As used in the description of the present invention, the terms “insulinprecursor” or “pro-insulin” are described as a single-chain polypeptidein which, by one or more subsequent chemical and/or enzymatic processes,may be converted into human insulin.

As used in the description of the present invention, the term“pro-insulin analog” is defined as a pro-insulin molecule having one ormore mutations, substitutions, deletions, and or additions, of the A, Band/or C chains relative to the native human pro-insulin nucleic acidsequence. The pro-insulin analogs are preferably such wherein one ormore of the naturally occurring nucleic acids have been substituted withanother nucleic acid within a triplet encoding for a particular aminoacid.

The term “a” as used in the description of the present invention isintended to mean “one or more”, and is used to define both the singularand plural forms of the item or items to which it references, or to afeature or characteristic to which it refers. The use of the singular orplural in the claims or specification is not intended to be limiting inany way and also includes the alternative form.

The term “about” is intended to be inclusive of and to encompass both anexact amount as well as an approximate amount or range of values orlevels of the item, ingredient, element, activity, or other feature orcharacteristic to which it references. Generally, and in someembodiments, the term “about” is intended to reference a range of valuesrelatively close to the specific numerical value specificallyidentified. For example, “about 3 grams to about 5 grams” is intended toencompass a measure of in or around a value of 3 grams, concentrationvalues between 3 grams and 5 grams, concentration values in and around 5grams, as well as concentration values that are exactly 3 grams andexactly 5 grams.

As used in the description of the present process, a high proteinconcentration of the pro-insulin or insulin product is defined as aprotein yield concentration of at least about 3 grams/liter, or betweenabout 3 grams to about 5 grams per liter. The expression yield to beexpected may be defined as a protein/peptide yield that is sufficient todetect via polyacrylamide gel electrophoreses (PAGE).

The invention in a general and overall sense relates to an improvedprocess for preparing a heterologous recombinant protein in aprokaryotic host cell. This process is characterized in that it employsa unique recombinant protein that provides a useful and efficientlyprocessed pro-insulin peptide having a unique, modified C-peptideregion, as well as a His tagged N-terminal sequence.

By heterologous protein is meant that said protein in said prokaryotichost cell is not native, i.e., it occurs as a protein in peculiar orforeign (i.e., not native to) the host prokaryotic cell.

“Recombinant” means produced or modified by molecular-biologicalmethods.

As used in the description of the present invention, the term“heterologous recombinant protein” is defined as any protein known tothe skilled person in the molecular biological arts, such as, forexample, insulin, pro-insulin, C-peptide, and proteins containing thesetogether with any other protein or peptide fragment.

Prokaryotic host cells may be any host cells known to the skilledartisan in the molecular biological arts, and by way of example,Escherichia coli. Such types of cells available form public collectionsand useful in the practice of the present invention include, by way ofexample, the Deutsche Sammlung von Mikrooganismen and Zellkulturen GmbH,raunschweig, Germany, e.g., E. coli Strain K12 JM107 (DSM 3950).

Proteins and peptides are chains of amino acids linked by peptide bonds,which in the case of proteins give a defined structure that is typicallyrequired for activity. Peptides are chains of amino acids which may ormay not have activity or a defined structure.

Human Insulin Amino Acid sequence: The sequence of amino acids whichmake up the native insulin A and B chains is as follows:

(SEQ ID NO: 3) FVNQHLCGSHLVEALYLVCGERGFFYTPKTGIVEQCCTSICSLYQLENY CN

The following reference Table 1 provides the triplet codonscorresponding to each of the various amino acids that are used in thedescription of the present invention. As will be understood by those ofskill in the art, the amino acid that may be used in any particularlydefined position as part of any of the peptide, protein, or constructsotherwise defined herein by reference to a particular nucleotide tripletbase pair may be encoded by a number of different nucleotide tripletsthat function to encode the same amino acid. For example, where theamino acid of the sequence defined herein is alanine (Ala, or A), thetriplet codon of nucleic acids that may encode for this amino acid are:GCT, GCC, GCA, or GCG. The following table illustrates this definitionof variables at and substitutions as can be applied to all of thenaturally occurring amino acids sequences of the disclosure.

TABLE 1 U C A G U UUU UCU UAU UGU U {close oversize brace} Phe {closeoversize brace} Tyr Cys UUC UCC UAC UGG C UUA UCA {close oversize brace}Ser UAA Stop UGA Stop A {close oversize brace} Leu UUG UCG UAG Stop UGGTrp G C CUU CCU CAU CGU U {close oversize brace} His CUC CCC CAC CGC CCUA {close oversize brace} Leu CCA {close oversize brace} Pro CAA CGA{close oversize brace} Arg A {close oversize brace} Gln CUG CCG CAG CGGG

It should be understood that process steps within the followingdescription of the method may be modified, changed and/or eliminated,depending on the particular preferences of the processor and/or theparticular mechanical apparatus available to the processor, as well asthe specific reagents and/or materials available and/or convenienceand/or economics of use.

Example 1 General Materials and Methods

The present example describes some of the general techniques used in thepreparation and purification of the human pro-insulin product and in thefurther processing of the pro-insulin into a human insulin product.

A general outline of one method, by way of example and not exclusion, toisolate and/or enrich recombinant insulin from a composition that is notenriched for recombinant insulin and/or includes pro-insulin can bedescribed by the following series of steps:

1. Fermentation of E. coli transformed with the vector containing thehuman pro-insulin derivative-encoding amino acid sequence.

2. Lysis—Lyse the E. coli cells containing inclusion bodies enrichedwith the desired peptide, resuspended in a basic Tris/salt buffer, usinga Niro Soavi homogenizer.

3. Inclusion Body Washing—Contaminant protein removal is thenaccomplished via two sequential washes with a Tris/Triton X-100 buffer,followed by two sequential washes with a Tris/Tween-20 buffer, andfinally a single wash with a Tris/NaCl buffer.

4. Solubilization—Inclusion bodies are then solubilized in 8M ureacontaining reducing agents. Complete solubilization is achieved byadjusting the pH to 10.5 with NaOH.

5. Dilution refolding—The solubilized protein is then diluted intorefolding buffer (5 mM CAPS, pH 10.5 at 4° C.) to a final concentrationof 0.5 mg/ml. Allow the sample to refold for ≧48 hours at 2-10° C. Addan equal amount of oxidized glutathione to the initial amount ofreducing agent used in the solubilization buffer, followed by 5M NaCland 1M Phosphate additions, to final concentrations of 250 mM and 25 mMrespectively. Adjust pH to 7.9 with 6M HCl.

6. IMAC Chromatography—Load the dilute pro-insulin derivative containingcomposition onto an IMAC column to a maximum capacity of ≦15 mg/ml ofresin. Elute the pro-insulin via a 15CV gradient from 0-400 mMImidizole. Using RP-HPLC for analysis, pool the appropriate fractionscontaining the Pro-Insulin peak of interest at the desired purity level.

7. Buffer exchange—To the pool, add EDTA to a final concentration of 10mM. Exchange the buffer using a membrane with a suitable molecularweight cutoff (ex. 3000 Da). The final buffer should be at least 97%exchanged to a 20 mM Tris-C1, pH 8.0 at 2-10° C. A protein concentrationof approximately 10 mg/ml is desirable. Just prior to tryptic digest, 1MGlycine stock (pH 9.3-9.7 cold) is added to a final concentration of 100mM and the sample pH is adjusted to 9.7 (cold).

8. Initial Trypsin Enzymatic Transformation/Proteolysis—The bufferexchanged sample is digested with a 2000:1 mass ratio of protein totrypsin. Once complete, based on HPLC, the digest is then quenched bythe addition of acetic acid to ≧700 mM, to a pH of approximately 3.5.HPLC of the digest should show about 54% R30 and DI-R(30&31) insulinanalogs.

9. Reverse Phase Chromatography—The digested insulin is loaded onto aC18 column and eluted isocratically using a buffer of 23% acetonitrile,200 mM Sodium Sulfate and 0.16% phosphoric acid. Alternatively, a C4column may be used with a 22% acetonitrile, 200 mM Sodium sulfate and0.16% phosphoric acid buffer.

10. Buffer Exchange—Exchange the buffer using a membrane with a suitablemolecular weight cutoff (3000 Da). The final buffer should be at least97% exchanged to 5 mM acetic acid. 1M Glycine stock (pH 9.3-9.7 cold) isthen added to a final concentration of 100 mM, which shifts the pH ofthe sample to approximately 8.6. The pH is then adjusted toapproximately 9.3 with NaOH, and the sample is concentrated to 8-12mg/ml.

11. Carboxypeptidase B transformation—The buffer exchanged sample isdigested with a 1:1000 ratio of protein to carboxypeptidase B. Thedigest is monitored by RP-HPLC to determine reaction completion.

12. Crystallization—To the carboxypeptidase B digested insulin, an equalvolume of crystallization buffer (2.4M NaCl, 0.1M Citric acid, 6 mM ZincChloride) is added, pH adjusted to ˜6.3, and the sample is incubated atroom temperature. Completion of crystallization is determined by UVanalysis of the supernatant. Insulin crystals are harvested bycentrifugation or filtration, washed with ethanol, and dried in vaccuo.When ready for use, the recombinant product will be solubilized andportioned into appropriate sized individually packaged units. Forexample, the insulin prepared according to the present invention may beprepared in 100 unit/ml vials.

Example 2 Process for Preparation of Human Pro-Insulin Derivative in aModified ptrcHis 2A (Kan) Vector

The present example demonstrates one of the expression vectors that maybe used in the preparation of an appropriate vector that may be used totransform an appropriate cell capable of expressing the humanpro-insulin derivative. The specific vector described here is theptrcHis2A vector. This ptrcHis2A vector was first modified before thehuman pro-insulin derivative-encoding nucleic acid sequence was insertedinto the vector.

The ptrcHis2A vector may be purchased from a commercial vendor (e.g.,Invitrogen). Such a vector will then be modified to include a Kanamycinresistance gene in the middle of the ampicillin resistance gene so as tonegate the ampicillin resistance. Ampicillin resistance heightens thepotential for allergic reactions to preparations made using vectorconstructs that include the ampicillin resistance gene. Therefore it ispreferable to eliminate the ampicillin resistance in the constructs thatare prepared and used.

Example 3 Construction of Purified Human Pro-Insulin Gene Segment forInsertion into Vector

The present example is presented to demonstrate an example of the stepsof a process that may be used in the present invention for preparing thehuman pro-insulin derivative nucleic acid sequence, as well as forpreparing the modified C-peptide construct disclosed herein. The nucleicacid segment isolated in the present example was used as the startingmaterial for creating the various insertion nucleic acid sequencesdescribed in the following examples.

The nucleic acid sequence of ATCC deposited clone, MCG-12292, wasidentified by the present inventors to include a nucleic acid sequencethat encoded the native human pro-insulin gene. The human pro-insulingene sequence was isolated from the nucleic acid sequence of the ATCCdeposited clone, MCG-12292, and employed as a starting material in thepreparation of the various modified forms of human pro-insulin andpro-insulin derivatives having the mini-C-peptide sequence substitutionas defined herein.

Human Pro-insulin Gene in ATCC Clone MGC-12292: (Nucleic acid Sequenceof interest=nucleic acid sequence at positions 132-392 (Pro-Insulin) ofthe clone). The initial pDNR-LIB vector containing the nucleic acidsequence of interest was isolated/purified from the MCG-12292 cloneusing a QIAprep Spin Miniprep Kit.

The following nucleic acid sequence is a portion of the nucleic acidsequence of the ATCC Clone MCC-12292 that was identified and selected bythe present inventors, and does not represent the entire nucleic acidsequence of the deposited clone.

(SEQ ID NO: 4)   1agccctccag gacaggctgc atcagaagag gccatcaagc agatcactgt ccttctgcca  61tggccctgtg gatgcgcctc ctgcccctgc tggcgc tgct ggccctctgg ggacctgacc 121cagaggcagc  ctttgtgaac caacacctgt gcggctcaca cctggtggaa gctctctacc 181tagtgtgcgg ggaacgaggc ttcttctaca cacccaagac ccgccgggag gcagaggacc 241tgcaggtggg gcaggtggag ctgggcgggg gccctggtgc aggcagcctg cagcccttgg 301ccctggaggg gtccctgcag aagcgtggca ttgtggaaca atgctgtacc agcatctgct 361ccctctacca gctggagaac tactgcaact agacgcagcc cgcaggcagc cccccacccg 421ccgcctcctg caccgagaga gatggaataa agcccttgaa ccaacaaaaa aaaaaaaaaa 481aaaaaaaaaa aaaaa

The following nucleic acid sequence is a portion of the nucleic acidsequence of the ATCC Clone MGC. The portion of the nucleic acid sequencethat is bolded in the above sequence represents an amino acid fragment,which is not required in the final pro-insulin molecule. The underlinedportion represents the sequence fragment of interest (nucleic acids132-392 of SEQ ID NO:4). The sequence is as follows:

(SEQ ID NO: 5)   1tttgtgaac caacacctgt gcggctcaca cctggtggaa gctctctacc tagtgtgcgg  60gsaacgaggc ttcttctaca cacccaagac ccgccsggag gcagaggacc tgcaggtggg gcaggtggag130ctgggcgggg gccctggtgc aggcagcctg cagcccttgg ccctggaggg gtccctgcag aagcgtggca200 ttgtggaaca atgctgtacc agcatctgct ccctctacca gctggagaac tactgcaact ag

With the use of PCR, the above sequence of interest (nucleic acids132-393 of SEQ ID NO:4) was amplified with an additional leader sequence(atgcatcatcatcatcatcatgaaggtggccgc (SEQ ID NO:34)=new start codon,histidine tag, and tryptic cleavage sequence) and purified using aQIAprep PCR purification kit.

Translation: The amino acids below depict the original native sequencecoded in the ATCC Clone MGC-12292.

(SEQ ID NO: 6) MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICS LYQLENYCN

The initial cloning step removed the bolded amino acid region above andreplaced it with the new leader sequence in bold below. The “GGR”represents the tryptic cleavage site, which will be utilized in thetryptic transformation reaction to remove this new leader sequence.

(SEQ ID NO: 7) MHHHHHHGGRFVNQHLCGSHLVEALYLVCERGFFYTPKTRREAEDLGVGGVELGGGPGAGSLGPLALEGSLQKRGIVEQCCTSICSLYQLENYCNThe underlined amino acids represent the C-peptide region of the nativehuman pro-insulin molecule.

Cloning Procedure

The initial cloning step into the pTrcHis2A(Kan) vector utilized theEcoR1 site for the C-terminal ligation position, and the Nco1 site,indirectly, for the N-terminal ligation. In order to use an Nco 1 sitedirectly for an N-terminal ligation, the gene of interest must containan amino acid residue at position 2, which is encoded by a codon thatstarts with a guanine nucleotide. As the construct of interest does nothave the required amino acid at position two, a “blunt end” ligationreaction was utilized instead of a direct Nco1 “sticky end” ligation.

The sequence of the RBS site and the MCS of the ptrcHis2A(Kan) vector isas follows:

(SEQ ID NO: 8) RBS             NcoI          MCS            EcoRI3′ TAAGGAGGAATAAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTACCATATGGGAATTC 5′ (SEQID NO: 35)3′ TAAGGAGGAATAAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTATATGGGAATTC 5′

Primer Design:

Forward Primer: The forward primer will introduce the new N-terminalHistidine Tag and tryptic cleavage site. The forward primer was orderedwith a phosphorylated 5′ end, which is required for the blunt endligation reaction.

(SEQ ID NO: 9) 5′ (Phosphorylated) catcatcatcatcatcat ggtggccgc tttgtgaaccaacacctgtgcggctc 3 ′

Reverse Primer: The reverse primer will introduce the EcoR1 site intothe C-terminus of the sequence. The EcoR1 site will be used toaccommodate the insertion of the Pro-insulin sequence into the vector.The reverse primer sequence is as follows:

(SEQ ID NO: 10)                             EcoRI3′ gatggtcgacctcttgatgacgttgatc-cttaagg 5′

The new generated PCR product is as follows:

(SEQ ID NO: 11) His Tag      GGR 5′-Phos-c atcatcatcatcatcatg gtggccgctt tgtgaaccaa cacctgtgcggctcacacct ggtggaagct ctctacctagtgtgcgggga acgggcttc ttctacacacccaagacccg ccgggaggca gaggacctgcaggtgggca ggtggagctg ggcgggggccctggtgcagg cagcctgcag cccttggccctggaggggtc cctgcagaag cgtggcattgtggaacaatg ctgtaccagc atctgctccctctaccagct ggagaactac tgcaactagt ccttaagg 3′              EcoR1

Following the PCR reaction, the insert DNA was purified using theQIAprep Spin Miniprep Kit from Quaigen. This purified insert DNA wasused to create a pTrcHis2A(Kan) vector having this purified Pro-insulingene sequence (See Example 4).

Example 4 Ligation Reaction for Cloning the N-Terminal ModifiedPro-Insulin Gene into the Modified pTrcHis2A (Kan) Vector

The present example demonstrates the process by which the Pro-insulingene was ligated into the modified pTrcHis2A(Kan) vector.

A 5′ blunt end and a 3′ EcoR1 ligation reaction were utilized to insertthe Pro-insulin gene into the pTrcHis2A (Kan) vector.

The sequence at the MCS site of the pTrcHis2A(Kan) is as follows:

(SEQ ID NO: 12)       RBS Site         NcoI5′ TAAGGAGGAATAAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTACCATATATGGGAATTC 3′                                                            EcoRI

Initial cut of the Vector with Nco1: 5′ TAAGGAGGAATAAAC 3′ (SEQ IDNO:13) Nco1 cut leaves a “CATG” 5′ overhang. 3′ ATTCCTCCTTATTTGCTAC 5′(SEQ ID NO:14) 5′ TAAGGAGGAATAAACCATG 3′ (SEQ ID NO:15) T4 DNApolymerase fills in the opposing strand 3 ATTCCTCCTTATTTGGTAC 5′ (SEQ IDNO:16) leaving a blunt end.

Blunt End Reaction: The DNA was digested with Nco1 for 1 hour at 37° C.using 2 μg of DNA and 10 units of Nco1. Following the hour digestion, 2units of T4 DNA polymerase were added to the reaction and incubated at12° C. for 15 minutes. The blunt end reaction was then stopped byaddition of EDTA to a concentration of 10 mM and heating to 75° C. for20 minutes. The vector DNA was then purified using the QIAprep SpinMiniprep Kit from Qiagen.

Following purification, the other end of the MCS was cut with EcoR1:

5′ TAAGGAGGAATAAACCATG (SEQ ID NO: 17) AATTC 3′3′ ATTCCTCCTTATTTGGTACTTAA (SEQ ID NO: 18) G 5′

Now that the vector was prepared, the insert was digested with EcoR1,leaving a blunt ended phosphorylated N-terminus and an EcoR1 sticky endon the C-terminus. Following the EcoR1 digestions, the vector and insertDNA were both purified using the QIAprep Spin MiniPrep Kit from Qiagen.

Once purified, the insert was ligated into the vector using a 4 to 1molar ratio of insert to vector DNA at 12° C. overnight.

Transformation: One microliter of the ligation reaction was used totransform competent BL21 E coli cells, which were plated on LB-Kan agarplates and incubated overnight at 37° C. Several clones were picked andsent to IUPUI for DNA sequencing. Clones with the correct sequence werescreened for expression. Good expression was verified in all clones.

Example 5 Site Directed Mutagenesis for the Conversion of Lysine 64 toAlanine in the C-Peptide Region of Human Pro-Insulin

A site directed mutagenesis PCR reaction was employed to convert thelysine at position 64 to alanine The constructed His-tagged-Gly-Gly-ArgPro-insulin gene constructed in Example 4 was used as the template.

The creation of this amino acid mutation eliminates the possibility ofgeneration Arg-AO-insulin during purification. Trypsin is an enzyme thathas specific cleavage activity at the C-terminus of arginine residues,and to a lesser extent, towards the C-terminus of lysine residues. Inthe transformation reaction, it is required that the C-peptide,including the arginine at position 65, be removed along with theN-terminal sequence. If trypsin cleaves at the lysine in position 64, itwill be unable to remove the arginine at position 65, due to the factthat it requires at least one residue on both sides of a cleavage site.What results is the production of an unwanted by-product,arg-A-0-insulin. This by-product constitutes a small loss in yield andgenerates an undesired contaminant.

By converting this lysine 65 into another uncharged amino acid,particularly alanine, the arg-AO-insulin product is not formed. This isbecause the trypsin no longer acts to cleave at this particular site ofthe pro-insulin sequence.

Site directed mutagenesis was used to convert the lysine at position 64to alanine. The procedure was adapted from the protocol in theStratagene Quick Change Site Directed Mutagenesis kit. The PCR reactionutilized pFU Turbo polymerase because of its high fidelity compared withTaq polymerase. Site directed mutagenesis involves the synthesis of theentire gene along with the vector (pTrcHis2A(Kan)). The insulin(Met-His-tagged-Gly-Gly-Arg)/)pTrcHis2A(Kan) clone synthesized above(Example 4) was used as the template for the PCR reaction.

PCR Primers Used:

Initial Sequence:

                     K64 5′ ccctggaggggtccctgca gaagcgtggcattgtggaacaatgctgtacc 3′ (SEQ ID NO: 19) Forward Primer:5′ ggggtccctgcaggcgcgtggcattgtg 3′ (SEQ ID NO: 20) Reverse Primer:3′ ccccagggacgtccgcgcaccgtaacac 5′ (SEQ ID NO: 21)

The 50 μl PCR reaction was treated with 20 units of Dpn1, and incubatedat 37° C. for 1 hour to digest all methylated template DNA beforetransforming chemically competent BL21 cells.

Following transformation into BL21 cells, several clones were sequencedfor gene verification. The clone that was isolated was:Met-His-taggedGly-Gly-Arg/Pro-insulin/K64A/pTrcHis2A(Kan).

The expression of this clone was very good.

Final Gene product (Met-His tagged/Gly-Gly-Arg/Pro-Insulin/K64A):

(SEQ ID NO: 22) 5′ ATGCATCATCATCATCATCATGGTGGCCGCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACC CGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTT GGCCCTGGAGGGGTCTCTGCAGGCGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAG 3′

Amino Acid Sequence of the His-tagged/Gly-Gly-Arg/K64A Pro-insulin:

(SEQ ID NO: 230 MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAEDLGVGGVELGGGPGAGSLQPLALEGSLGAR GIVEQCCISICSLYQLENYCN

In the nucleic acid and amino acid sequences above, the bold andunderlined portions identify the connecting C-peptide sequence.

Example 6 Full Length Substituted Pro-Insulin Construct and Uses Thereofin Production of Human Insulin

With regards to the full length Pro-Insulin substitution construct ofHis-Tagged/K64A, three unique modifications are present to the originalgene that simplify purification and increase yield.

-   -   1) The N-terminal sequence was modified by the addition of a        6-Histadine tag, which could be used to simplify the        purification via the use of a Nickel chelating column as an        initial purification step following refolding.    -   2) Following the 6-His tag sequence on the N-terminus, a well        documented tryptic cleavage site was introduced in order to        provide a simple means of removing the N terminal tag following        the metal chelating chromatography step. The sequence introduced        was “Glycine, Glycine, Arginine”, with cleavage after the        arginine.    -   3) The third modification was the conversion of an amino acid        located at position 64 (original Pro-insulin) of the native        sequence, which is a lysine residue that is converted to an        alanine The modification prevents the formation of        arg-AO-insulin during the tryptic cleavage transformation step,        which increases the theoretical yield.

All data thus far supports the modifications. The initial chelatingcolumn step yields a Tagged-Pro-Insulin pool of approximately greaterthan or equal to about 92% purity. The tryptic transformation stepyields a final insulin molecule with high digestion efficiency whichdemonstrates the effectiveness of the “gly, gly, arg” cleavage sequencefor N-terminal removal and the lack of an Arg-A-0-insulin at position 65demonstrate the advantage of the replacement at position 64.

The individual transformation reactions, rather then a singletransformation reaction, allows for the efficient removal of theDesthreonine byproduct which is created by cleavage at the Lysine atposition 29. This cleavage can be minimized in the native sequence byintroducing nickel to the transformation reactions. However, thepresence of the Histidine tag in the clone described herein, preventsthis nickel protection of lysine 29. The separate transformationreactions allow for almost complete removal of the desthreoninebyproduct, which is created when trypsin cleaves at the lysine atposition 28, removing the threonine at position 29.

The final purification steps, including the Reverse phaseChromatography, carboxy peptidase B transformation reactions andcrystallization, yields a highly pure insulin (≧99%) sample, which byHPLC reverse phase analysis shows essentially no Pro-insulin analogs orN-terminal fragments (See FIG. 2). As well, the desthreonine contaminantcan essentially be completely removed.

Amino Acid Sequences

The natural pro-insulin amino acid sequence is as follows:

(SEQ ID NO: 25) MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSIC SLYQLENYCN

Replacement of initial sequence with Tag and cleavage site:

(SEQ ID NO: 26) MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

Conversion of lysine 64 to alanine:

(SEQ ID NO: 27) MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCN

Example 7 Biopotency Study of the Full Length Substituted Pro-InsulinConstruct

The final purified Insulin samples were tested in vivo on rabbits tocompare the biopotency against Humulin R. As analytical studies give asimple picture of a biological molecule's relative state compared with astandard, it is imperative that a bioassay be used to determine that thepurified molecule of interest carries the required biological activity.

In Vivo Biological Method

The biological assay was on the current accepted procedure outlined inthe United States Pharmacopeia. Testing was conducted by MPI Research.

Test subjects included 30 male rabbits which were given injections ontwo separate days of either the control (saline), positive control(Humulin R), or test sample (IPT (Elona Biotechnologies) Human insulin),through subcutaneous injection.

Dosing levels consisted of 0.35 or 0.7 international units (IU),administered at a dose volume of 0.35 ml/dose. The control groupreceived saline on both days 1 and 3 at a dose volume of 0.35 ml/dose.

Glucose monitoring was conducted prior to dosing and at 30, 60, 90, 150,and 240 minutes following dosing on both days. Results showed comparablebiopotency of IPT human insulin with Humulin R (see FIG. 3).

Example 8 Pro-Insulin Constructs

The present example demonstrates the utility of the present inventionfor providing unique Pro-Insulin constructs that are particularly usefuland efficient in the methods of insulin production described herein.

Native Pro-Insulin AA sequence: recombinant Lys/Pro insulin and/orincludes pro-insulin can be described by the following series of steps:

1. Fermentation of E. coli transformed with the vector containing thehuman pro-insulin derivative-encoding amino acid sequence:

2. Lysis—Lyse the E. coli cells containing inclusion bodies enrichedwith the desired peptide, resuspended in a basic Tris/salt buffer, usinga Niro Soavi homogenizer.

3. Inclusion Body Washing—Contaminant protein removal is thenaccomplished via two sequential washes with a Tris/Triton X-100 buffer,followed by two sequential washes with a Tris/Tween-20 buffer, andfinally a single wash with a Tris/NaCl buffer.

4. Solubilization—Inclusion bodies are then solubilized in 8M ureacontaining reducing agents. Complete solubilization is achieved byadjusting the pH to 10.5 with NaOH.

5. Dilution refolding—The solubilized protein is then diluted intorefolding buffer (5 mM CAPS, pH 10.5 at 4° C.) to a final concentrationof 0.5 mg/ml. Allow the sample to refold for ≧48 hours at 2-10° C. Addan equal amount of oxidized glutathione to the initial amount ofreducing agent used in the solubilization buffer, followed by 5M NaCland 1M Phosphate additions, to final concentrations of 250 mM and 25 mMrespectively. Adjust pH to 7.9 with 6M HCl.

6. IMAC Chromatography—Load the dilute pro-insulin derivative containingcomposition onto an IMAC column to a maximum capacity of ≦15 mg/ml ofresin. Elute the pro-insulin via a 15CV gradient from 0-400 mMImidizole. Using RP-HPLC for analysis, pool the appropriate fractionscontaining the Pro-Insulin peak of interest at the desired purity level.

7. Buffer exchange—To the pool, add EDTA to a final concentration of 10mM. Exchange the buffer using a membrane with a suitable molecularweight cutoff (ex. 3000 Da). The final buffer should be at least 97%exchanged to a 20 mM Tris-C1, pH 8.0 at 2-10° C. A protein concentrationof approximately 20-25 mg/ml is desirable.

8. Trypsin and Carboxypeptidase Enzymatic Transformation—The bufferexchanged sample is digested with a 2000:1 and 1000:1 mass ratio ofprotein to trypsin and protein to carboxypeptidase B respectively. Oncecomplete, based on HPLC, the digest is then quenched by the addition ofacetic acid to ≧700 Mm, to a pH of approximately 3.5. HPLC of the digestshould show about 54% Humalog (Lys/Pro insulin).

9. Reverse Phase Chromatography—The digested Lys/Pro insulin is loadedonto a C18 column and eluted isocratically using a buffer of 23%acetonitrile, 200 mM Sodium Sulfate and 0.16% phosphoric acid.Alternatively, a C4 column may be used with a 22% acetonitrile, 200 mMSodium sulfate and 0.16% phosphoric acid buffer.

10. Buffer Exchange—Exchange the buffer using a membrane with a suitablemolecular weight cutoff (˜3000 Da). The final buffer should be at least97% exchanged to 0.01N acid, and the sample is concentrated to 8-12mg/ml.

11. Crystallization—To the Lys/Pro insulin. an equal volume ofcrystallization buffer (2.4M NaCl, 0.1M Citric acid, 6 mM Zinc Chloride)is added, pH adjusted to ˜6.3, and the sample is incubated at roomtemperature. Completion of crystallization is determined by UV analysisof the supernatant. Insulin crystals are harvested by centrifugation orfiltration, washed with ethanol, and dried in vaccuo. When ready foruse, the recombinant product will be solubilized and portioned intoappropriate sized individually packaged units. For example, the insulinprepared according to the present invention may be prepared in 100unit/ml vials.

The present example demonstrates several advantages that utilization ofthe Lys/pro insulin construct has over the original insulin sequenceused in the purification scheme seen Example 1:

-   -   a. Step 7 does not require the Glycine addition and pH        adjustment to 9.7, which decreases the chances of desamino        formation, typically seen in the high or low pH ranges.    -   b. The separate digestion reaction found in steps 8 and 11 of        Example 1 are combined into a single digestion reaction in Step        8 above, which is carried out at pH 8.0, which decreases the        possibility of desamino formation.    -   c. The Lys/pro insulin construct prevents the formation of        Desthreonine-insulin, which is created in the trypsin        transformation reaction. It represents approximately a 6-10%        yield loss, and can only be separated from the Arg and Di-Arg        insulin species on the reverse phase step.

Example 10 Sodium Sulfate in Recovery Process for Insulin

The reverse phase chromatography step for purification of Di-Arg andSingle-Arg insulin species following tryptic digestion and prior tocarboxypeptidase B digestion involves one of two methods:

1. A shallow gradient elution from 23.5% to 25% acetonitrile in thepresence of 200 mM sodium sulfate and 0.16% phosphate, over 15 columnvolumes.2. An isocratic elution of 23.5% acetonitrile in the presence of 200 mMsodium sulfate and 0.16% phosphate, over approximately 15 columnvolumes.

The above methods are specific to a C 18 reverse phase system with a 15μM particle size and 200-300 Å pore, but may be adapted to a C4 or C8system by adjusting the acetonitrile concentrations. As well, theparticle size may be varied to decrease back pressure.

A study of the above methods was conducted to look at the effects ofsodium sulfate on the chromatography. It was found that the sodiumsulfate when removed from the buffers resulted in a high yield loss. Thematerial did not seem to stick to the column effectively, leading tosome material crashing off at the column void volume. Sodium sulfate isrequired to increase mass transfer within the column. (See FIGS. 5A and5B).

This study demonstrates the requirement of sodium sulfate in the reversephase buffers. Although a more detailed study has not been conducted todetermine the minimum required concentration, it is also understood thatthe flow rate during loading is a crucial parameter, since the slowerthe load, the better chance that there will be binding to the columnbefore the void volume is through.

All of the compositions and methods disclosed and claimed herein can bemade and expressed without undue experimentation in light of the presentdisclosure.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the compositions, themethods, and in the steps and in the sequence of steps of the methodsand processes described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutions and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinventions disclosed herein by the appended claims. Accordingly, theexclusive rights sought to be patented are as described in the claimsbelow.

BIBLIOGRAPHY

The following references are specifically incorporated in theirentirety.

-   1. U.S. Pat. No. 6,777,207 B2—Kjeldsen et al. (2004).-   2. U.S. Pat. No. 4,916,212—Markussen et al. (1990).-   3. U.S. Pat. No. 5,962,267—Shin et al. (1999).-   4. EP Patent No. 0 055 945—Goeddel et al. (1981).-   5. Chance et al. (1981), Peptides: Proceedings of the 7^(th)    American Peptide Chemistry Symposium, pp. 721-728 (Rich, D. and    Gross, E. eds.).-   6. Chan et al. (1981), P.N.A.S., U.S.A., 78:5401-5404.-   7. Thim et al. (1986), P.N.A.S., U.S.A., 83: 6766-6770.-   8. Frank et al. (1981), Peptides: Proceedings of the 7^(th) American    Peptide Chemistry Symposium, pp. 729-739 (Rich, D. and Gross, E.    eds.).-   9. Chang et al. (1998), Bio hern J. 329: 63 1-635.

1. A nucleic acid sequence encoding a polypeptide comprising thesequence A-RREAEALQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO:32)-B, whereinsaid A chain and said B chain are native human insulin chains.
 2. Anucleic acid sequence encoding the polypeptideRREAEALQVGQVELGGGPGAGSLQPLALEGSLQA (SEQ ID NG:32) R.


3. An expression vector comprising the nucleic acid sequence of claim 1.4. The expression vector of claim 3, wherein the expression vector isPro-insulin (HisTagged)/K64A/pTrcHis2A(Kan),His:tagged-Gly-Gly-Arg/Pro-insulin/(Kan). 5.A microorganism transformed with the vector of claim
 4. 6. Themicroorganism of claim 5 further defined as an E. coli transformed withplasmid Pro-Insulin (His Tagged)/K64A/pTcHis2A(Kan) plasmid.
 7. An aminoacid sequence comprising RREAEALQVGQVELGGGPGAGSLQPLALEGSLQA(SEQ ID NG:32) R.


8. A composition enriched for a peptide encoded by the amino acidsequence of claim
 7. 9. A process for preparing a composition enrichedfor an insulin analog employing a modified human pro-insulin peptidecomprising: (a) preparing an isolated nucleic acid sequence encodingnative human pro-insulin; (b) modifying said nucleic acid sequence byproviding a molecular tag on said modified pro-insulin sequence so as toprovide a histidine-tagged modified pro-insulin peptide; (c) convertinga lysine residue to an alanine residue at position 64 in a C-peptideregion of said pro-insulin sequence to provide a modified pro-insulinderivative gene sequence; (d) inserting the modified pro-insulinderivative gene sequence into a suitable vector to provide a vectorcomprising said modified pro-insulin derivative gene sequence; (e)transfecting a culture of competent cells comprising E. coli cells withsaid vector to provide transformed E. coli cells; (f) culturing thetransformed E. coli cells under conditions suitable for expression ofthe modified pro-insulin derivative gene sequence; (g) disrupting saidpopulation of transformed E. coli cells to provide a compositioncomprising inclusion bodies containing the modified human pro-insulin;(h) solubilizing said composition to provide a composition comprisingunfolded peptide; (i) refolding said unfolded peptide to providerefolded human pro-insulin derivative peptide; (j) passing saidcomposition over a metal chelating column (Nickel chelate) to purifysaid composition and collecting a purified preparation of said refoldedhuman pro-insulin derivative peptide; (k) transforming the collectedpurified preparation of refolded human pro-insulin derivative peptideinto Arg and Di-Arg peptide species by tryptic digestion; (l) purifyingsaid Arg and Di-Arg peptide species by passing said peptides through areverse phase column; and (m) transforming the purified Arg and Di-Argpeptide species into insulin by carboxypeptidase B digestion andharvesting an insulin analog.
 10. The process of claim 9 wherein the E.coli is a BL21 production E. coli.
 11. The process of claim 9 whereinthe peptide is further defined as comprising an amino acid sequenceencoding a substituted human pro-insulin derivative protein having anamino acid sequence comprising: (SEQ ID NO: 30)MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQARGIVEGCCTSICSLYGLENYCN.


12. The process of claim 9 wherein the reverse phase chromatographycolumn is further defined as comprising a silica based media with a C4,C8, or C18 bonded phase.
 13. The process of claim 9 wherein the suitablevector is a pTrcHis2A (Kan) vector.
 14. A pharmaceutical productcomprising recombinant human insulin prepared by the process of claim 9.15. A process for preparing a human pro-insulin derivative comprising aC-peptide RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO:33), saidprocess comprising: (a) preparing a pro-insulin peptide having aC-peptide amino acid sequence RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQA(SEQ ID NG:33) R;

(b) modifying said peptide to include a met-histidine-Gly-Gly-Arg tag atthe N-terminus of said peptide; (c) incorporating said nucleic acidsequence into an appropriate vector to provide a transformed vector;(pTrcHis vector); (d) transforming a population of competent cellscomprising E. coli cells with said vector to provide transformed E. colicells; (e) selecting transformed E. coli cells that express a peptidecomprising an amino acid sequence -RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR(SEQ ID NO:33); (f) culturing a composition of said selected transformedcells under conditions suitable for expression of the peptide havingsaid amino acid sequence; (g) solubilizing said composition comprisingsaid cells; and (h) purifying the human pro-insulin derivative from theculture.
 16. The process of claim 15 further comprising the step: (i)crystallizing the human pro-insulin derivative.
 17. The process of claim15 further comprising preparing human insulin from the human pro-insulinderivative by enzymatic hydrolysis.
 18. The process of claim 15 whereinthe E. coli is a BL21 production E. coli.
 19. A composition prepared bythe process of claim 17, wherein said composition is essentially free ofhuman pro-insulin.